PROJECT SUMMARY Staphylococcus aureus is responsible for a large number of hospital- and community-acquired infections worldwide. The rise in the incidence of S. aureus infections is primarily due to a combination of increased antibiotic resistance and augmented virulence of strains associated with community infections. In the absence of a protective vaccine, studies aimed at dissecting virulence strategies of S. aureus are desperately needed with the hope of identifying novel targets for the generation of new treatments to combat this pathogen. An important pathogenic strategy of S. aureus is the production of exotoxins that target and kill host cells. Among these, S. aureus strains associated with human infections can produce up to five different pore-forming bi- component toxins known as leukocidins. The long-term objective of our program is to understand the molecular details by which these leukocidins influence the pathophysiology of S. aureus infection through targeting cells of the immune system. The present application focuses on one of these toxins as a model leukocidin, leukocidin ED (LukED). The importance of the proposed research originates from our recent discoveries that LukED: (i) is a critical virulence factor involved in the lethality of mice upon S. aureus bloodstream infection, (ii) is required for promoting bacterial replication in vivo, (iii) contributes to S. aureus pathogenesis by targeting and killing immune cells in vivo, and (iv) targets a wide array of host cells in a receptor-dependent manner. The goals of this research program are to understand the mechanisms by which LukED targets its different host receptors, to define the details by which LukED injures endothelial cells to promote the lethality associated with bloodstream infection, and to elucidate how LukED interacts with the other leukocidins to modulate pathogenesis. To this end, we propose to employ a multidisciplinary approach that combines toxin-receptor biochemical studies with primary human cell biology and novel murine models of infection. The data gathered from these studies will provide much needed insight into the molecular details of how the bi-component leukocidins contribute to S. aureus pathobiology.